Sarcocystis neurona is an apicomplexan parasite that is the primary cause of equine protozoal myeloencephalitis (EPM; Dubey et al., 1991), which is a common and debilitating infectious disease that affects the central nervous system of horses. S. neurona is related to the human and animal pathogen Toxoplasma gondii and to the important veterinary pathogen Neospora spp. The geographic range of S. neurona appears to be limited to the western hemisphere, thus EPM primarily affects horses in the Americas.
Definitive antemortem diagnosis of EPM remains exceedingly difficult, for a variety of reasons. Horses afflicted with EPM exhibit signs that are similar to a number of different neurological disorders (MacKay et al., 2000). Furthermore, S. neurona infection does not equate to disease, since only a small proportion of seropositive horses will suffer from EPM (MacKay et al., 2000); as a consequence, the detection of anti-S. neurona antibodies in serum provides little diagnostic information other than indicating previous exposure to the parasite. Analysis of cerebrospinal fluid (CSF) to reveal intrathecal antibody production has improved the predictive value of antibody detection for EPM diagnosis. However, interpretation of CSF antibody presence can be confounded by contamination of the CSF sample with minute amounts of serum antibodies (Miller et al., 1999).
Other contemporary diagnostic assays provide only mediocre predictive value for EPM diagnosis. Western blot analysis (a.k.a., immunoblot) of crude S. neurona lysate remains the principal immunodiagnostic test that is used to detect antibodies in suspect EPM horses (Granstrom et al., 1993). The assay relies on the recognition of several antigens, primarily in the low molecular weight range, by serum/CSF antibodies (Dubey et al., 2001b; Granstrom et al., 1993; MacKay et al., 2000). Unfortunately, Western blot analysis is primarily a research tool that is relatively laborious and somewhat hindered by subjectivity, so any improvements to the immunoblot are of limited value. While the immunoblot has been utilized for a number of years to help diagnose EPM, it is a first-generation test that needs to be replaced with Improved assays based on simplified, and thus more reliable, techniques that are more appropriate for diagnostic use.
Nucleic acid amplification assays (polymerase chain reaction; PCR) for S. neurona detection have been developed based on the S. neurona ribosomal RNA genes (Fenger et al., 1994; Marsh et al., 1996). These PCR-based assays detect the presence of S. neurona DNA, and therefore the parasite, in the horse, so they can provide a definitive indication of active infection. However, prior to the present invention, these nucleic acid-based tests have been inherently unreliable. Specifically, parasites may be very few or non-existent in a CSF sample, so there will be few or no available target molecules (i.e., parasite genomic DNA) for PCR amplification. More importantly, the general use of PCR for diagnosis is still suspect. Although measures can be taken to improve the reliability of PCR, the technique continues to be troubled by both false positive and false negative results.
The selection of an antigen for development of a diagnostic test can be somewhat subjective since any particular pathogen is composed of numerous antigenic proteins. Logically, the target molecule in a diagnostic assay must elicit a detectable antibody response in the infected animal. In this regard, surface antigens of the Coccidia, such as the primary surface antigens of Toxoplasma gondii (Handman and Remington, 1980; Sharma et al., 11983) and Neospora caninum (Howe et al., 1998), are exceedingly immunogenic. These surface antigens have been designated SAGs and SAG-related sequences (SRSs). Significantly, the TgSAG1 surface antigen of T. gondii has been shown to protect mice against acute toxoplasmosis (Bulow and Boothroyd, 1991), and the NcSAG1 (p29) major surface antigen of N. caninum has been used to develop an ELISA for detection of Neospora infection in cattle (Howe et al., 2002). Collectively, these previous studies demonstrate that coccidian SAGs are at least candidate proteins for the development of both diagnostic assays and protective vaccines.
Despite the foregoing art, prior to the present invention it had not been shown that the surface antigens of S. neurona (i.e., SnSAG2, SnSAG3, and SnSAG4) are effective target molecules for examining immune responses in infected horses and for developing improved assays for EPM diagnosis. Such molecules would also provide the basis for improved vaccines and diagnostic kits, including antigen and antibody kits, for fast and reliable diagnosis of S. neurona infection.